The objective of this proposal is to develop a quantitative ELISA assay for quantitative measurement of anti-RhD antibodies. Specific aims include isolation of soluble serologically active RhD, coating ELISA plates with the extract, enhancing the sensitivity of antibody detection using the Photochemical Amplification Method (PAM) and the Fluorescence Enhancement Technique (FET), measuring stability of extract after drying, and analyzing the IgG subclass distribution of anti- RhD antibodies. Soluble RhD extract will be coated on ELISA plates according to the patented protocols with varying concentrations of extract, ELISA assays using PAM and FET will be optimized and standard curves for quantitation of anti-RhD antibodies will be constructed with standards of known anti-RhD concentration. Studies of anti-Rh subclass distribution will be done after ELISA optimization. Successful completion of the project will provide protocols for eliminating variations in the current doubling dilution titering method by making available a strictly quantitative means of measuring anti- RhD antibodies. Measurement of anti-RhD subclasses in patient sera will provide new insight into known variations in severity of Hemolytic Disease of the Newborn. The research will create new business opportunities and will benefit patients. PROPOSED COMMERCIAL APPLICATION: This research will lead to development of protocols for producing commercial kits for quantitative ELISA measurement of anti-RhD antibodies. These kits would be used in Blood Banks and Pathology Labs for measuring anti-RhD antibody levels in pregnant women who are carrying an RhD positive fetus. Such antibodies mediate the disease Hemolytic Disease of the Fetus. ELISA measurement would standardize assays eliminating the known variation in reported results from current methods.